Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M.

In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).


INTRODUCTION
Aromatic hydrocarbons such as BTEX (benzene, toluene, ethylbenzene, and xylenes) which are found in crude oil, are used in many industrial products and known to be pollutants. They can seep through the soil and groundwater from underground storage tanks and pipeline leakages or waste disposal practices and contaminate water and groundwater [1]. The Environmental Protection Agency (EPA) classified BTEX as priority pollutants and revealed their toxic effects on living organisms and environments [2]. Reducing and cleaning up hydrocarbons from the environment is thus considered to be a very important process, and the remediation of contaminated sites has received special attention. Compared with physical or chemical methods, microbial degradation is one of the most effective and economical solutions for the removal of aromatic hydrocarbons from contaminated environments [3]. Microbial enzymes such as hydrocarbon dioxygenase can be valuable for such biodegradation processes. Aromatic hydrocarbon dioxygenases have been shown to have an important role in the biodegradation of BTEX by the intradiol or extradiol cleavage of the aromatic ring. These enzymes are identified in many bacteria such as Rhodococcus, Pseudomonas, and can degrade hydrocarbons to less toxic compounds such as catechol [4,6].
Since the benzene ring cleavage is essential for the aromatic hydrocarbon degradation process, benzoate and its derivatives are common compounds that can be produced through aerobic and anaerobic hydrocarbon biodegradation [3]. Under aerobic conditions, the ring is cleaved by adding oxygen through an oxidation reaction; hence, different intermediate compounds such as arene cis-diols could be obtained [7,8]. The production of cis-diols by enzymes such as toluene dioxygenase, naphthalene dioxygenase and biphenyl dioxygenase which are known as Rieske type non-heme iron oxygenases, has been previously reported [9]. During the next step, catechol can be produced and the reaction that follows can lead to Kerbs cycle intermediates. The aerobic degradation of benzoate has been elucidated in various bacteria [3]. Benzoate dioxygenase from Acinetobacter calcoaceticus ADP1 and P. putida have been shown to catalyze the dihydroxylation of benzoates [5,10,11].
Many genes such as xyl, bop and ben are involved in the dihydroxylation benzoate ring, encode various dioxygenases by plasmids or chromosomes, and yield a nonaromatic cis-diol to be converted into catechol [6]. The benA gene is involved in benzoate metabolism in many Rhodococcus strains [12]. Because of their high catabolic diversity for hydrocarbon degradation, Rhodococcus species are frequently isolated from contaminated environments and widely used in bioremediation [3]. In Malaysia, Rhodococcus ruber UKMP-5M was isolated from oil contaminated soil, whereas other studies showed the bacterium to be capable of degrading crude oil and toluene [13]. The activity of catechol dioxygenase shows that R. ruber UKMP-5M can degrade catechol by catechol 2,3 dioxygenase using a meta pathway to produce 2-hydroxymuconic acid [14]. The present study focused on the expression, purification and characterization of benzoate dioxygenase from Rhodococcus ruber UKMP-5M. The results can help characterize a functional gene for the bioremediation of polluted environments.

MATERIALS AND METHODS
DNA amplification and cloning: Total DNA from Rhodococcus ruber UKMP-5M was extracted by a Wizard genomic DNA purification kit (Promega) and analyzed using 1% agarose gel and a spectrophotometer. A specific primer was designed based on genome sequences from R. ruber UKMP-5M with two restriction sites BamHI and HindIII to amplify the benA gene in an automated thermal cycler (Bio-Rad). We used the Forward 5-'GGA TCC ATG GAA CTG GAC CGA GGT CCA GCA GGT GC-3' and Reverse:5'-AAGCTTTCAGAAGGGGTAGACGGGCACGTCGCG-3' primers.
The PCR product (~ 0.6) was purified using a QIA Miniprep purification kit (Qiagen). The purified benA fragment was ligated into a pGEM ® -T Easy vector by T4 DNA ligase (Promega) and transformed into competent cells of E. coli DH5α after being heat shocked at 42ºC for 50s. The cells were grown on LB agar containing ampicillin (Sigma) (50 µg/ml), 50 mg/mL 5-bromo-4-chloro-3-indoyl-β-D-Galactopyranoside (X-Gal) (Promega) and 100 mM isopropyl β-D-1-thiogalacto pyranoside (IPTG) (Sigma), and the positively transformed E. coli was screened by PCR. The recombinant plasmid pGEMT-benA was extracted via QIAprep Miniprep kit (Promega) and analyzed using 1% agarose gel. The pGEMT-benA and pET 28b vector (Novagen) were digested using BamHI and HindIII (Promega) and the purified fragment benA was inserted into pET 28b by T4 DNA ligase after incubation at different temperatures (14-16°C) for 14-17 hours. The product was then transformed into E. coli DH5α using heat shock, the cells were cultured on LB agar supplemented with kanamycin (50 µg/mL), and the recombinant plasmid pET 28b-benA was extracted. The DNA sequences of pGEMT-benA and pET 28b-benA were evaluated using specific benA and universal primers and the data were analyzed using BLAST and VecScreen bioinformatics software.

Growth of recombinant E. coli:
The pET 28b-benA was transformed into E. coli BL21 (DE3). A pre-culture from the transformed E. coli was prepared and incubated at 37ºC to reach optical density ~ 0.5 at OD550nm. The standard inoculum (10%) was added to Minimal salt medium (MSM) [13] supplemented with (0.5-2.5 mM) sodium benzoate. The culture was incubated at 30°C, 150 rpm for 72 hours and the growth was measured at OD550nm. The control was prepared with the same condition as E. coli but without the plasmid.

Expression and purification:
A single colony of E. coli BL21 (DE3) containing plasmid pET 28b-benA was inoculated into 10 mL LB broth containing 50 µg/mL kanamycin and incubated at 37ºC, 250 rpm for 16 hours. The culture was centrifuged at 4ºC, 4000 rpm for 15 minutes and the pellet was diluted 5-fold (50 mL) with pre-warmed LB broth. The cells were incubated with shaking to obtain an OD550nm of 0.5-0.7. Afterward, 1 mL of the culture was collected as a non-induced sample and the medium was cooled to 22ºC to limit the formation of inclusion bodies. The expression of benzoate dioxygenase was induced by adding 0.5 mM IPTG and taking it out after 2, 4, 6 and 16 hours. The cells were harvested by centrifugation at 13000 rpm, 4°C for 20 minutes. The pellet was washed and resuspended in a lysis buffer (50 mM NaH2PO4 with 300 mM NaCl) (pH 8.0) containing 1 μg/mL lysozyme, and 0.1 mM/mL phenylmethyl sulfonyl fluoride (PMSF) (Sigma) was added to protect the protein structure. The mixture was then incubated in ice for 30 min. Cell disruption was performed by a sonicator (Sonics-vibra cell) at 20s pulses with 5 minutes' rest for 6 times. The suspension was centrifuged at 4°C, 12000 rpm for 40 minutes. The resulting supernatant and pellet were analyzed by SDS-PAGE. The expression of His-tag protein was confirmed by western blot analysis using a monoclonal antibody (Antibody Histidine-Alkaline Phosphatase) (Sigma). The over expression of benzoate dioxygenase was carried out in a large scale (at least 2L) and the supernatant was filtered using a 0.22 µm membrane filter. The protein was purified by ion exchange chromatography (IXC) using an AKTA prime plus system (No 1314455 Sweden) (GE Health care) according to the manufacturors' instructions. The machine and Hi Trap SP-FF Sepharose column (i.d×h 0.7×2.5 cm) were washed stepwise with buffer A (wash buffer) which contained 50 mM acetic acid (pH 4.3) or a 50 mM HEPES buffer (pH 8.0) at a flow rate of 0.8 mL/min. The crude protein was eluted in buffer B (elution buffer) containing 200 mL washing buffer with 0-1.0 M NaCl supplemented with 0.5-1 mM dithiothreitol (DTT) or mercaptoethanol (ME). In the last step, the machine and column were re-equilibrated with buffer A. The collected fractions from bound and unbound proteins were analyzed using SDS-PAGE and western blotting to determine the molecular weight and purity of the target protein. The concentration of benzoate dioxygenase was determined using a Bicinchoninic acid (BCA) kit at OD562nm. After purification, the desired protein was concentrated by a vivaspin or Amicon (Millipore) protein column to the maximum volume of 3 mL and kept at -20ºC.

Enzyme characterization:
The reaction of benzoate dioxygenase was measured by converting reduced β-Nicotinamide adenine dinucleotide (NADH) to β-Nicotinamide adenine dinucleotide (NAD) by a spectrophotometer at 340nm. All assays were carried out in triplicates. The reaction mixture was prepared as follows: 50 mM sodium/potassium phosphate (110 µL), 0.1 mM FeSO4 (11 µL), sodium benzoate as a substrate (1.1 µL), 2 mM reduced β-Nicotinamide dinucleotide phosphate (3.5 µL) and purified benzoate dioxygenase (100 µL).The reaction mixture was adjusted to 1.1 mL by adding H2O (784.4 µL). The reaction was monitored at 1 minute intervals for 10 minutes at 25°C by sa pectrophotometer at 340 nm. The enzyme activity was calculated using a general equation based on unit/mL, where one unit is the amount of enzyme that catalyzes the conversion of 1.0 µmol of substrate to the expected product per minutes at a standard assay condition [15].
Optimal pH and temperature: Optimum pH was determined in standard assay conditions as described above, except that the following buffer systems from 6.0 to 11.0 were used: 50 mM sodium/potassium phosphate (pH 6-7.5), Tris (hydroxylmethyl) methylamine (pH 8-9.5) and NaHCO3 (pH 10-11) at 0.5 intervals. The mixture was incubated for 3 minutes at 25ºC and OD340nm was measured. At optimal pH, the temperature was adjusted to 4, 20, 25, 37, 40, 50, 60, 70 and 80°C for the reaction mixture. The enzyme activity was measured after 3 minutes of incubation by spectrophotometry at OD340nm.

Determination of kinetic parameters:
The effect of substrate concentration on enzyme activity was determined by maximum velocity (Vmax) and Michaelis constant (Km) by varying the concentration of sodium benzoate at a range of 0.1-1.2 mM to the total adjusted volume of 1.1 mL using water. The mixture was incubated for 3 minutes at 25°C and NAD production was measured at OD340nm. The Km and Vmax were calculated using Lineweaver Burk plots.
Substrate specificity: Different substrates (benzene, benzoic acid, butyl benzene and ethyl benzene) at 1 mM concentration were replaced with sodium benzoate and the efficiency of each substrate for benzoate dioxygenase activity was measured at OD340nm.

Analysis of metabolites:
The mixture was prepared in a total volume of 1 mL and the reaction was stopped after 5 minutes of incubation by adding 300 µL of 0.1 M HCl. Protein was removed from the mixture using vivaspin 500 (Sartorious, Germany) to add 300 µL diethyl ether. One microliter from the upper layer was injected into the injection port of the gas chromatography-mass spectrometry (GC-MS) system [16].

RESULTS AND DISCUSSION
Rhodococcus ruber UKMP-5M has the ability to degrade many aromatic hydrocarbons including toluene and catechol [14]. In this research, benzoate dioxygenase from R. ruber UKMP-5M was purified and the kinetic characteristics were determined. Benzoate and its derivatives were degraded via hydroxylation by incorporating two oxygen atoms into the aromatic nucleus [17].
The benA gene from R. ruber UKMP-5M was amplified at 64°C, yielding a product of 0.6 kb. The recombinant plasmid pGEMT-benA was 3.6 kb. Sequence analysis of the benA fragment showed 70% similarity with the aromatic ring dioxygenase using Rhodococcus equi 103S. The pGEMT-benA plasmid was digested to two fragments; the linear pGEMT fragment (3000 bp) and the inserted fragment benA (600 bp). Plasmid pET 28b-benA was constructed successfully into E. coli DH5α after incubation for 14 hours at 15°C. The recombinant pET 28b-benA was  6 kb which was included in pET 28b (5.4 kb) and the benA fragment (0.6 kb). The accuracy of the benA fragment was confirmed by PCR amplification and sequencing analysis.
The highest growth of E. coli BL21 containing pET 28b-benA was determined when cells were incubated in 0.5 mM sodium benzoate for 24 hours, as compared to 1 and 2.5 mM of sodium benzoate. In Klebsiella oxytoca C302 the highest growth at 0.5 mM benzoate occurred after 3 hours of incubation [18]. The highest growth in Rhodococcus sp LIN was observed after 33 hours of incubation in 2 mM benzoate [3]. For S. marcescens DS-8, the highest growth was observed after incubation in 5 mM of sodium benzoate for 72 hours [19].
The transformed E. coli BL21 (DE3) was induced with 0.5 mM IPTG at 22°C, 250 rpm after 6 hours. The molecular weight of benzoate dioxygenase was 25 kDa (Figure 1a, b lane 2) and the isoelectric point (pI) was 10.38. The obtained protein was purified and eluted with an NaCl concentration of 1.0 M that showed into flow-through fractions 11-12 of column. The purified benzoate dioxygenase was observed as a single band on the SDS-PAGE and western blot (Fig.1a,b lane3). The concentration of purified benzoate dioxygenase was 1.71 mg/mL, purified at 8 folds with a 62% yield by ion exchange chromatography (Table 1)  The highest benzoate dioxygenase activity was 6.54 U/mL, which was close to the benzoate dioxygenase activity by P. putida (6.67 U/mL). The benzoate dioxygenase activity by P. arvilla C-l was 4.2 U/mg [20]. The reported benzoate dioxygenase activities of A. calcoaceticus ADP (18.2 U/mg) [5], P. putida ML2 (20 U/mg) [21], and P. putida sp (58 U/mg) [15] were higher than that of R. ruber UKMP-5M. The benzoate dioxygenase activity of Amycolatopsis species such as A. rugosa strains DSM 43194, DSM 43387 and DSM 43388, was in the range of 12-53 U/mg [22], which is slightly higher than what was observed in the present study. Similar to other studies, the cofactor NADH and exogenous Fe +2 were necessary for the benzoate dioxygenase activity of R. ruber UKMP-5M [15,23,24]. A marked concentration-dependence for crude and purified benzoate dioxygenases demonstrated that benzoate dioxygenase activity increased when the reaction mixture was incubated with successive portions of substrate [15].
The optimal pH for benzoate dioxygenase activity was found to be 8.5 (Figure 2 a), which was close to the finding of other studies regarding Arthrobacter and Rhodococcus strains [25]. The optimal pH of S. marcescens DS-8 [19] and Hydrogenophaga (Betaproteobacteria) was 8.0 [20]; however, the optimum pH for K. oxytoca C302 [25] and P. arvilla C1 [18] were 7.0 and 6.7, respectively, which were both less than the optimal pH for the benzoate dioxygenase activity of R. ruber UKMP-5M. For R. ruber UKMP-5M, the optimal temperature for benzoate dioxygenase activity was 25°C and the enzyme activity decreased considerably at any temperature lower than 20°C or higher than 60°C (Fig. 2b). The optimal temperatures for benzene dioxygenase activities of K. oxytoca C302 and P. arvilla C1 were 30°C and 24°C, respectively [18,25]. The Vmax and Km values for benzoate dioxygenase of R. ruber UKMP-5M were 7.36 U/mL and 5.58 µM, respectively. The km for benzoate dioxygenase of A. calcoaceticus ADP1 (26 µM) [26] and P. putida (32 µM) [5] were much higher than the Km value of R. ruber UKMP-5M, but the Km value of P. arvilla C1 (3 µM) was less than R. ruber UKMP-5M [27].
Benzoate dioxygenase has a role in many hydrocarbon pathways because it can be used in various substrates [12]. The recombinant benzoate dioxygenase from R. ruber UKMP-5M was able to utilize benzene, ethyl benzene and benzoic acid as well as sodium benzoate (Fig.2c). Several studies have reported that B. subtilis strain MO5, Rhodococcus rhodochrous strain DSM 43274T, Pseudomonas stuzeri 11C2 [21], Rhodococcus sp. strain DK17 [24] and A. calcoaceticus ADP1 [5] used similar substrates. However, higher enzyme activity was observed by the benzoate substrate [5]. Identification of reaction products: The end metabolite of the reaction was cyclohexamine dione that was observed in GC-MS. In the Rhodococcus strain, 19070 benzoate was converted to non-aromatic cis-diol [28]. The benzene dioxygenase of P. putida ML2 catalyzed the double hydroxylation of benzene to produce cis-1, 2dihydroxycyclohexa-3, 5-diene [21]. The benA gene was able to cleave with the benzene ring in substrates such as benzene and sodium benzoate; however, several genes were needed to produce catechol [29].
The amino acid sequence of benzoate dioxygenase from R. ruber UKMP-5M showed a 53% similarity with benzene 1, 2 dioxygenase of P. putida F1 and a 60% similarity with Mycobacterium vanbaalenii PYR-1. The homology for benzoate dioxygenase R. ruber UKMP-5M with biphenyl dioxygenase from P. putida F1 and terminal dioxygenase R. erythropolis were 47% and 60%, respectively. The benzoate dioxygenase family showed more sequence similarity with aromatic dehydrogenases such as biphenyl and toluene dioxygenase [30,31]. Benzoate dioxygenase oxidizes aromatic hydrocarbons and related compounds by hydroxylationg the dioxygenase ring to catalyze dioxygenation reactions [32,33]. R. ruber UKMP-5M benzoate dioxygenase functions in different pathways where the benA gene assists other genes to convert benzoate to catechol. The results of our study indicate that this bacterium has the ability to biodegrade other BTEX, because the benzoate dioxygenase was able to use benzene and ethyl benzene as substrate. These abilities suggested that R. ruber UKMP-5M possesses an aromatic ring-hydroxylating dioxygenase similar to other aromatic hydrocarbon dioxygenases in Pseudomonas and Acinetobacter encoded by xyl or ben genes.